Brainstem catecholaminergic neurons induce torpor during fasting by orchestrating cardiovascular and thermoregulation changes

Abstract

Torpor, an adaptive hypometabolic state in response to fasting, is characterized by pronounced reductions in body temperature, heart rate, and thermogenesis. However, how the brain orchestrates these physiological changes to induce torpor and the relationships among them remain elusive. Inhibiting catecholaminergic (CA) neurons in the ventrolateral medulla (VLM) significantly impairs torpor in mice, while their activation reduces body temperature, heart rate, energy expenditure, physical activity, and thermogenesis. Importantly, the heart rate decline precedes body temperature reduction, resembling patterns observed in natural torpid animals. Moreover, a likely causal relationship exists between heart rate reduction and body temperature decline. VLM-CA neurons may regulate heart rate and thermogenesis through projections to the dorsal motor vagal nucleus and medial preoptic area, respectively. Additionally, these neurons are conserved in Daurian ground squirrels and become active before hibernation, indicating their potential role in hibernation. Here, we find that VLM-CA neurons play important roles in fasting-induced torpor.

Fig. 1. VLM-CA neurons are activated by fasting and required for fasting-induced torpor.

a Experimental strategy for telemetric recording of core body temperature and locomotor activity. b The core body temperature (T_core) and locomotor activity of a non-fasting mouse (upper panels) and a fasting mouse (bottom panels). c–e Fasting induced Fos (green) expression in tyrosine hydroxylase positive (TH⁺) VLM-CA neurons. c Representative images from mice at different time points during fasting along the rostral-caudal axis. Arrows indicate Fos positive (Fos⁺) CA neurons. Scale bars: 100 µm. d The percentages of Fos⁺ neurons among VLM-CA neurons at different time points. e The percentages of Fos⁺ neurons among VLM-CA neurons along the rostral-caudal axis. Control (n = 3 mice), 6 h (n = 5 mice), 9 h (n = 4 mice) and 15 h (n = 4 mice) in (d, e). f–h Chemogenetic inhibition of VLM-CA neurons. f The viral strategy. g Representative images of n = 12 mice showing the expression of hM4Di (red) in VLM-CA neurons. h Representative electrophysiological trace showing CNO (50 µM)-induced inhibition of hM4Di⁺ VLM-CA neurons. Changes in the core body temperature (T_core) (i) and locomotor activity (j) of Dbh^VLM-hM4Di and Dbh^VLM-mCherry mice during 24-h food deprivation. k Quantitative analysis of the T_core. Changes in the average T_core before and after CNO injections (left), variance (middle), and lowest value (right) of the T_core during fasting. l Total torpor time spent with T_core ≤ 31 °C and number of torpor bouts with T_core ≤ 31 °C during fasting. m Quantitative physical activity during the periods of 10–18 h and 10–24 h post food deprivation. The dashed lines in (i, j) denote the timing of the CNO injections. The gray bar indicates the dark phase (20:00 to 8:00). Two-tailed unpaired Student’s t-test in (d, k, l, m). Two-tailed paired Student’s t-test in (e). Two-way ANOVA analysis in (i). ns, not significant (P > 0.05). Data are presented as the means ± SEM. Source data are provided as a Source Data file. Figure 1a was created in BioRender. Zhan, C. (2025) https://BioRender.com/s14s282. Figure 1f was created in BioRender. Zhan, C. (2025) https://BioRender.com/fi02cic.

Fig. 2. VLM-CA neurons are sufficient to induce torpor in non-fasted mice.

a–c Chemogenetic activation of VLM-CA neurons. a The viral strategy. b Representative images of n = 5 mice showing the expression of hM3Dq (red) in TH⁺ VLM-CA neurons. Scale bars: 100 µm. c Representative electrophysiological trace showing CNO (10 µM)-induced activation of hM3Dq⁺ VLM-CA neurons. d–h Changes in the core body temperature (T_core) (d, P < 0.0001) and physical activity (f, P = 0.0057) of Dbh^VLM-hM3Dq and Dbh^VLM-mCherry mice following CNO or saline injections. e Quantitative analysis of changes in the average T_core (left), variance (middle) and lowest (right) T_core after CNO or saline injections. g Quantitative physical activities. h Total torpor duration and number of torpor bouts. The dashed lines in (d, f) denote the timing of CNO or saline injection. Two-way ANOVA analysis in (d, f). Two-tailed paired Student’s t-test in (e, g, h). Data are presented as mean ± SEM. ns, not significant P > 0.05. Source data are provided as a Source Data file. Figure 2a was created in BioRender. Zhan, C. (2025) https://BioRender.com/fi02cic.

Fig. 3. Activation of VLM-CA neurons reduces metabolic rate and thermogenesis.

Oxygen consumption (a), carbon dioxide production (b), and calculated energy expenditure (c) in Dbh^VLM-hM3Dq and Dbh^VLM-mCherry mice following CNO or saline injections. Quantitative data for the 6 h following CNO or saline injections are shown in the bottom panels. d–f iBAT thermogenesis of Dbh^VLM-hM3Dq mice following CNO or saline injections. d Experimental setup. e Representative infrared images showing surface body temperature at 0, 30, 60, and 120 min after CNO or saline injections. f Quantitative analysis of the iBAT surface temperature. g–i iBAT sympathectomy. g Representative image of iBAT sympathectomy, involving cutting off five sympathetic nerve bundles. Noradrenaline (NA) concentration (h) and representative images (i, n = 3 mice) of sympathetic nerves (red, TH) in iBAT after sympathectomy or sham surgery. Scale bar: 500 µm. Relative mRNA expression of Ucp1 gene in iBAT (j), T_core (k), iBAT temperature (l), and locomotion activity (m) of Dbh^VLM-hM3Dq mice before and after sympathectomy following CNO and saline injections. Two-way ANOVA analysis in (a, b, c, f, k, l, m). Two-tailed unpaired Student’s t-test and paired Student’s t-test in the bottom panels of (a, b, c, h), as appropriate. One-way ANOVA in (j). Data are presented as mean ± SEM. ns not significant P > 0.05. Source data are provided as a Source Data file. Figure 3d was created in BioRender. Zhan, C. (2025) https://BioRender.com/fi02cic.

Fig. 4. Activation of VLM-CA neurons enhances heat dissipation and promotes cool preference.

a–c Optogenetic activation of VLM-CA neurons. a The viral strategy. b Representative images showing the expression of SSFO (green) in TH⁺ VLM-CA neurons. Scale bar: 100 µm. c Representative electrophysiological trace illustrating the activation of SSFO⁺ VLM-CA neurons by a blue light pulse (2 s, blue bar). d–g The body temperature of Dbh^VLM-SSFO and Dbh^VLM-GFP control mice before and after light stimulation (indicated by blue bars, 20 Hz, 30 s). Representative infrared images (d), quantitative analysis of tail (e) and iBAT (f) surface temperature, and core body temperature (g, P = 0.0033). h–j Thermal gradient assay schematic with heatmaps depicting the cumulative positions of a representative Dbh^VLM-hM3Dq mouse on a thermal gradient for 50 min following saline or CNO injection. The average gradient position (0 = 50 °C, 1 = 15 °C) (i, P = 0.0386) and median gradient position (j, P = 0.0003, each point represents one mice) during the 50-min period. The dashed lines in (i) denote the timing of CNO or saline injection. Two-way ANOVA analysis in (e, f, g, i). Two-tailed paired Student’s t-test in (j). Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001. Source data are provided as a Source Data file. Figure 4a was in created in BioRender. Zhan, C. (2025) https://BioRender.com/k4e71f1. Figure 4h includes a mouse illustration created in BioRender. Zhan, C. (2025) https://BioRender.com/k4e71f1.

Fig. 5. Activation of VLM-CA neurons triggers preceding heart rate decline prior to body temperature drop.

a–c ECG recordings of heart rate in freely behaving Dbh^VLM-hM3Dq mice. a Experimental setup for ECG recordings. b Heart rate of freely behaving Dbh^VLM-hM3Dq mice after CNO or saline injection (P < 0.0001). c Representative ECG traces in mice 1 h after saline or CNO injections. Red arrows indicate skipped heartbeats. d Dynamic changes in heart rate and T_core in a WT mouse during fasting-induced torpor. The bottom panel shows the four-phase fluctuations during the period indicated by the dashed box in the upper panel. e–i Simultaneous recordings of heart rate and T_core in Dbh^VLM-SSFO mice following light stimulation (indicated by blue bars, 20 Hz, 30 s). e Experimental setup (left panel) and representative ECG traces (right panel). The red arrow indicates skipped heartbeats. f Changes in heart rate and T_core of Dbh^VLM-SSFO mice. g Latency to decrease (upper panel) and latency to the lowest points (bottom panel) of heart rate and T_core (n = 6 mice, each point represents one mice). h, i Representative traces of a Dbh^VLM-SSFO mouse showing the dynamic changes in heart rate and T_core after light stimulation. i The four-phase fluctuations during the period indicated by the dashed box in (h). Two-way ANOVA analysis in (b). Two-tailed paired student’s t-test in (g). Data are presented as mean ± SEM. ****P < 0.0001. Source data are provided as a Source Data file. Figure 5a, e were created in BioRender. Zhan, C. (2025) https://BioRender.com/fi02cic.

Fig. 6. Downstream neurons of VLM-CA in DVC and MPA regulate body temperature.

a, b Anterograde tracing of VLM-CA neurons. a Schematic of anterograde tracing (upper) and labeled VLM-CA neurons (bottom). b Representative images of VLM-derived projections in DVC, DMH, MPA, and PVN (n = 4 regions per 7 independent biological replicate). Scale bars: 200 µm. c Representative images of Fos signals in DVC, DMH, MPA, and PVN of Dbh^VLM-hM3Dq (bottom panels, n = 3 animals) and Dbh^VLM-mCherry control (upper panels, n = 2 animals) mice. Fos immunostaining was performed 2 h after CNO injections. Scale bars: 100 µm. Schematic of the Fos-Triggered Downstream Neuronal Activation (FTDNA) strategy (d) and representative results (e) of selective expression of SSFO in VLM-activated neurons (Fos⁺) in the MPA (n = 521 cells in 3 MPA area sections from 3 animals). FTDNA-based optogenetic activation (indicated by blue bars, 20 Hz, 30 s) of the VLM^M3 → PVN^SSFO (f, P = 0.0028), VLM^M3 → DMH^SSFO (g, P = 0.0012), VLM^M3 → MPA^SSFO (h, P = 0.0042), and VLM^M3 → DVC^SSFO (i, P = 0.0019) neural circuits, respectively. The left panels show SSFO expression and fiber implantation, and the right panels show T_core. Two-way ANOVA analysis in (f, g, h, i). Data are presented as mean ± SEM. *P < 0.05; **P ≤ 0.01. Source data are provided as a Source Data file.

Fig. 7. Functional roles of the VLM^Dbh → DVC and VLM^Dbh → MPA neural circuits in body temperature and heart rate regulation.

a, b Selective activation of the VLM^Dbh → DVC or VLM^Dbh → MPA neural circuits by direct infusion of CNO (1 µl, 0.5 µg/µl dissolved in saline) into the DVC or MPA through pre-implanted cannulas. a Experimental schematic. b Representative images of cannula positions in the DVC and MPA. The effects of selective activation of the VLM^Dbh → MPA neural circuit on core body temperature (c, P = 0.0461), heart rate (d, P = 0.0315), and iBAT thermogenesis (e, P = 0.0095). The effects of selective activation of the VLM^Dbh → DVC neural circuit on core body temperature (f, P = 0.0195), heart rate (g, P = 0.0084), and iBAT thermogenesis (h, P = 0.0122). i In vitro whole-cell recording of DMV neurons during optogenetic activation of VLM-CA neuronal projections (blue bar, 5 ms) in the DVC of Dbh^VLM-ChR2 mice. Representative traces (middle panel) of EPSCs following administering: (1) artificial cerebrospinal fluid (ACSF), (2) picrotoxinin (PTX), (3) 6,7-dinitroquinoxaline-2,3-dione (DNQX), and (4) after a washout period. Group data (right panel, n = 9 neurons). All recordings were performed during the application of TTX (10 µM) and 4-AP (1 mM). The effects of saline or isoproterenol on the heart rate (j) and T_core (k). l–r The impacts of selective inhibition of the MPA or DVC on the body temperature and heart rate. l Schematic of the FTDNI strategy. Changes in core body temperature (m, P = 0.1453), heart rate (n, P = 0.8122) and iBAT temperature (o, P = 0.3244) when selectively inhibiting MPA neurons. Changes in core body temperature (p, P = 0.7215), heart rate (q, P = 0.7967) and iBAT temperature (r, P = 0.9174) when selectively inhibiting DVC neurons. Two-way ANOVA analysis in (c–h, m–r). Two-tailed paired Student’s t-test in (i). Data are presented as mean ± SEM. ns, not significant P > 0.05; *P < 0.05; **P ≤ 0.01. Source data are provided as a Source Data file. Figure 7a includes a gavage tube illustration created with BioRender.com. Zhan, C. (2025) https://BioRender.com/k4e71f1. Figure 7i was created in BioRender. Zhan, C. (2025) https://BioRender.com/fi02cic. Figure 7l were manually drawn based on anatomical features using Microsoft PowerPoint.